There is growing evidence that the clustering and spatial pattern of T cell receptors is critical for T cell responses. Changes in clustering and pattern can alter cell responsiveness from low to high, or even to unresponsive. We aim to investigate the genetics and the biophysics of T cell receptor clustering and pattern. Genetic screens will be used to identify pathways that are important for the enhanced clustering of T cell receptors found on activated T cells. We will use an shRNA screen to focus on pathways known to affect T cell receptor clustering -glycosylation and the role of cholesterol and its interaction with the cytoskeleton. Second we will analyze the impact of genes identified in the screen in binding of a TCR ligand, soluble peptide-MHC-lg complexes to T cells and in vitro and in vivo T cell responses. In addition we will engineer quantum dots that will present TCR ligands, MHC/peptide or anti-CD3 to naive and activated T cells. The quantum dots will be used to probe the lateral organization of TCR on naive and activated T cells. Blinking of bound quantum dots will be analyzed quantitatively to follow changes in receptor organization with time. Synergies with other projects: A specific aim of Project 2, is to analyze the TCR organization and SMAC formation in activated CD4 cells. Project 1's work on the genetic control of TCR clustering will be directly relevant to that project. Also, the nanoprobes and methods for characterizing TCR organization of this project will be used to probe the nano-organization of TCR on naive or activated CD4+ cells. In Project 3 quantum dots will be used to characterize changes in TCR clustering during induction of anergy in vivo. Quantum dots and genetic manipulation will also be used in collaboration with Dr. Desiderio Project 4 to characterize T cell receptor states in cells an clones lacking TFII-I or carrying a mutated form of the protein. There has already been strong interaction with Project 5 in designing selection/screening assays for genetic control of TCR clustering. In addition using the techniques developed in Specific Aim 1 we will analyze the effects of the genes identified in Project 5 on TCR clustering. Many immune responses, for example responses to parasites, to tumors and to some viruses, are often blunted because T cells respond to antigen by turning off, rather than by activating and attacking their targets. Understanding the arrangement of T cell antigen receptors that is most likely to trigger T cells, will lead to development of methods to manipulate the T cell surface to produce a strong, focused response, or, to suppress response, rearranging the receptors to limit autoimmune responses.